Bacterial keratitis is a costly and global problem that results in vision loss and blindness, and Serratia marcescens is a leading agent of community-acquired Gram-negative bacterial keratitis. S. marcescens also causes many hospital acquired infections including pneumonia, endocarditis, bacteremia and urinary tract infections that are commonly resistant to current antibiotics and whose outcomes are associated with significant morbidity and mortality. There is a lack of studies that investigate how S. marcescens genes contribute to ocular infections using isogenic mutant strains. The broad long-term objective of this research is to prevent vision loss following corneal infections caused by this organism. A better understanding of the mechanisms by S. marcescens virulence factors are regulated will allow for a novel approach to reduce tissue damage and corneal opacification that results from the expression of these factors. This new knowledge about specific pathways can be used to custom design novel anti-infectives. Establishing new therapeutic targets is becoming ever more important as bacteria continue to develop resistance to existing classes of antibiotics. Our overall specific hypothesis to be tested is that the transcription factor EepR/S is a critical virulence factor that controls expression of tissue damaging metalloprotease and hemolysin activities. Our preliminary data supports that the mutation of EepR/S in S. marcescens severely attenuates the pathogenesis of S. marcescens in an in vivo ocular model of keratitis. Our central hypothesis will be tested by accomplishing the following aims: Aim 1. Test the hypothesis that EepR/S, four metalloproteases and the ShlA hemolysin are required for S. marcescens cytotoxicity in vitro and ocular pathogenesis in vivo. Aim 2. Test the hypothesis that EepR and EepS regulate transcription of metalloprotease and hemolysin genes, and that EepR/S is in a regulatory pathway with other transcription factors (crp, hexS and pigP). PUBLIC HEALTH RELEVANCE: Ocular infections caused by bacteria are a common and costly problem in the United States and in both developed and developing nations abroad. Over 100,000 Americans live with vision loss due to corneal infections (keratitis) caused by dangerous bacteria. The bacterium Serratia marcescens is a frequent cause of serious and sometimes fatal hospital acquired infections and vision impairing community-acquired corneal infections. S. marcescens genes that facilitate vision-threatening corneal infections have not been studied to date. This study is designed to determine how S. marcescens is able to successfully infect and damage the cornea. Specifically, we will evaluate the role of secreted proteases, a pore-forming hemolysin, and a newly discovered genetic regulatory system. The answers to these questions will allow for the creation of new drugs to treat S. marcescens ocular infections and reduce infection-associated vision loss.